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Alternative splicing produces structural and functional changes in CUGBP2.

Suzuki H, Takeuchi M, Sugiyama A, Alam AK, Vu LT, Sekiyama Y, Dam HC, Ohki SY, Tsukahara T - BMC Biochem. (2012)

Bottom Line: In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms.The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan. suzuki-h@jaist.ac.jp

ABSTRACT

Background: CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons.

Results: The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.

Conclusion: Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

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Expression analysis of the alternative splicing of the ACTN1 and IR genes. (A) Schematic representation of the mutually exclusive splicing of the ACTN1 and IR genes. The genomic structure and alternatively spliced mRNAs of ACTN1 are shown in the left panel and those of IR in the right panel. Exons are indicated as black boxes with alternatively spliced exons depicted as gray boxes. Introns are indicated with a central narrow line. The arrow indicates the primer sites. (B) Expression analysis of ACTN1 and IR in P19 neural differentiation. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons as shown in Figure 2A. The right side shows the positions of the SM exon and NM exon products or exon 11 skipping and inclusion products. (C) Expression analysis of ACTN1 and IR in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons. The right side shows the positions of the alternatively spliced products. β-Actin is shown as a control in Figure 1. The relative amounts of each PCR product were estimated by densitometry. Total expression levels were normalized using Day 0 samples (B) or brain samples (C). The error bars indicate the standard error. The values under the gel images indicate the percentage of the SM type or IRA in total ACTN1 transcripts or IR transcripts.
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Figure 2: Expression analysis of the alternative splicing of the ACTN1 and IR genes. (A) Schematic representation of the mutually exclusive splicing of the ACTN1 and IR genes. The genomic structure and alternatively spliced mRNAs of ACTN1 are shown in the left panel and those of IR in the right panel. Exons are indicated as black boxes with alternatively spliced exons depicted as gray boxes. Introns are indicated with a central narrow line. The arrow indicates the primer sites. (B) Expression analysis of ACTN1 and IR in P19 neural differentiation. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons as shown in Figure 2A. The right side shows the positions of the SM exon and NM exon products or exon 11 skipping and inclusion products. (C) Expression analysis of ACTN1 and IR in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons. The right side shows the positions of the alternatively spliced products. β-Actin is shown as a control in Figure 1. The relative amounts of each PCR product were estimated by densitometry. Total expression levels were normalized using Day 0 samples (B) or brain samples (C). The error bars indicate the standard error. The values under the gel images indicate the percentage of the SM type or IRA in total ACTN1 transcripts or IR transcripts.

Mentions: CUGBP2 is a regulator of the alternative splicing of several transcripts, including ACTN1 [11] and IR [17]. The alternative splicing of ACTN1 and IR was therefore analyzed in adult mouse tissues and P19 cells. The ACTN1 gene has mutually exclusive exons, namely the smooth muscle (SM) exon and non-muscle (NM) exon (Figure 2A). The SM exon., as a percentage of NM and SM exons, was higher in neural differentiated P19 cells (28.6%) than in undifferentiated P19 cells (6.7%, Figure 2B). In the brain, 83.7% of ACTN1 transcripts contained the SM exon, while the NM exon was predominant in the kidney (SM exon: 26.6%) and liver (Figure 2C, SM exon: 7.6%). CUGBP2 has been suggested to promote the inclusion of the SM exon in prior work [11,12], suggesting that the elevated expression of CUGBP2 and inclusion of the ACTN1 SM exon may occur in the same cells and tissues.


Alternative splicing produces structural and functional changes in CUGBP2.

Suzuki H, Takeuchi M, Sugiyama A, Alam AK, Vu LT, Sekiyama Y, Dam HC, Ohki SY, Tsukahara T - BMC Biochem. (2012)

Expression analysis of the alternative splicing of the ACTN1 and IR genes. (A) Schematic representation of the mutually exclusive splicing of the ACTN1 and IR genes. The genomic structure and alternatively spliced mRNAs of ACTN1 are shown in the left panel and those of IR in the right panel. Exons are indicated as black boxes with alternatively spliced exons depicted as gray boxes. Introns are indicated with a central narrow line. The arrow indicates the primer sites. (B) Expression analysis of ACTN1 and IR in P19 neural differentiation. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons as shown in Figure 2A. The right side shows the positions of the SM exon and NM exon products or exon 11 skipping and inclusion products. (C) Expression analysis of ACTN1 and IR in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons. The right side shows the positions of the alternatively spliced products. β-Actin is shown as a control in Figure 1. The relative amounts of each PCR product were estimated by densitometry. Total expression levels were normalized using Day 0 samples (B) or brain samples (C). The error bars indicate the standard error. The values under the gel images indicate the percentage of the SM type or IRA in total ACTN1 transcripts or IR transcripts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3368720&req=5

Figure 2: Expression analysis of the alternative splicing of the ACTN1 and IR genes. (A) Schematic representation of the mutually exclusive splicing of the ACTN1 and IR genes. The genomic structure and alternatively spliced mRNAs of ACTN1 are shown in the left panel and those of IR in the right panel. Exons are indicated as black boxes with alternatively spliced exons depicted as gray boxes. Introns are indicated with a central narrow line. The arrow indicates the primer sites. (B) Expression analysis of ACTN1 and IR in P19 neural differentiation. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons as shown in Figure 2A. The right side shows the positions of the SM exon and NM exon products or exon 11 skipping and inclusion products. (C) Expression analysis of ACTN1 and IR in adult mouse tissues. Semi-quantitative RT-PCR was performed using primers to detect the alternatively spliced exons. The right side shows the positions of the alternatively spliced products. β-Actin is shown as a control in Figure 1. The relative amounts of each PCR product were estimated by densitometry. Total expression levels were normalized using Day 0 samples (B) or brain samples (C). The error bars indicate the standard error. The values under the gel images indicate the percentage of the SM type or IRA in total ACTN1 transcripts or IR transcripts.
Mentions: CUGBP2 is a regulator of the alternative splicing of several transcripts, including ACTN1 [11] and IR [17]. The alternative splicing of ACTN1 and IR was therefore analyzed in adult mouse tissues and P19 cells. The ACTN1 gene has mutually exclusive exons, namely the smooth muscle (SM) exon and non-muscle (NM) exon (Figure 2A). The SM exon., as a percentage of NM and SM exons, was higher in neural differentiated P19 cells (28.6%) than in undifferentiated P19 cells (6.7%, Figure 2B). In the brain, 83.7% of ACTN1 transcripts contained the SM exon, while the NM exon was predominant in the kidney (SM exon: 26.6%) and liver (Figure 2C, SM exon: 7.6%). CUGBP2 has been suggested to promote the inclusion of the SM exon in prior work [11,12], suggesting that the elevated expression of CUGBP2 and inclusion of the ACTN1 SM exon may occur in the same cells and tissues.

Bottom Line: In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms.The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan. suzuki-h@jaist.ac.jp

ABSTRACT

Background: CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons.

Results: The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure.

Conclusion: Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.

Show MeSH
Related in: MedlinePlus